Effect of Ursodeoxycholic Acid Administration on Biliary Lipid Composition and Bile Acid Kinetics in Cholesterol Gallstone Patients E N R I C O R O D A , MD, A L D O R O D A , PhD, C L A U D I A SAMA, MD, D A V I D E F E S T I , MD, G I U S E P P E M A Z Z E L L A , MD, R I T A A L D I N I , MD, and L U I G I B A R B A R A , MD

The effect o f ursodeoxycholic acid ( UDCA) on bile lipid composition and bile acid kinetics was evaluated in seven cholesterol gallstone patients following one month o f UDCA administration (12 mg/kg/day). UDCA administration induces a significant reduction in the cholesterol saturation index (SI). After UDCA treatment, UDCA becomes the predominant biliary bile acid while chenodeoxycholic, cholic, and deoxycholic acid are significantly reduced. UDCA pool significantly increases, and chenodeoxycholic, cholic, and total bile acid pools significantly decrease. The reduction in bile lithogenicity during UDCA administration suggests that UDCA may be useful for cholesterol gallstone treatment in m a n .

It is w e l l k n o w n t h a t c h e n o d e o x y c h o l i c acid (CDCA) induces desaturation of bile and dissolves cholesterol gallstones in man (1-8). Recent studies (9-11) have shown that the oral administration of ursodeoxycholic acid (UDCA), the 7-beta epimer of chenodeoxycholic acid, also induces these effects. According to these authors the following differences between C D C A and U D C A t r e a t m e n t exist: (1) U D C A seems to be effective at smaller doses than C D C A ; and (2) as far as concerns U D C A , desaturation of bile and dissolution of gallstones are not dose-dependent. The present investigations were carried out to study the effects of short-term administration of high doses of U D C A on the biliary bile acid patterns, bile acid kinetics, and fasting bile lipid cornFrom the Department of Gastroenterology, S. Orsola Hospital, and Institute of Chemistry " G . Ciamician," University of Bologna, Italy. Address for reprint requests: Dr. Enrico Roda, S. Orsola Hospital, Via Massarenti n. 9, 40100 Bologna, Italy.

position in s e v e n patients with cholesterol gallstones. MATERIALS AND M E T H O D S Seven patients (two males and five females; mean age, 44 years) with radiolucent gallstones in functioning gallbladders were studied before and one month after UDCA administration (12 mg/kg/day). The dose was given as a divided dose before breakfast, lunch, and dinner. No patients discontinued the treatment during the study period. Patients were not receiving any other drugs during the period of UDCA administration. None of the patients was overweight. All patients gave informed consent. Conventional liver function tests (alkaline phospharases, glutamic oxaloacetic and glutamic pyruvic transaminases, gammaglutamyltranspeptidase, serum bilirubin, prothrombin time) and serum lipid pattern were performed before and one month after the commencement of UDCA administration. Patients were treated in the outpatient department. UDCA was supplied in gelatine capsule form of 125 mg by Giuliani SpA (Milano, Italy), and purity was greater than 98% by gas-liquid chromatography (GLC).

Digestive Disease and Sciences, Vol. 24, No. 2 (February 1979)

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RODA ET A L UDCA administration was not suspended during bile acid kinetics studies. Each patient received a standard diet (calorie composition being: fats 25%, proteins 20%, carbohydrates 55%) for 15 days prior to commencement of the study. Procedure. [24-14C]chenodeoxycholic acid (radiopurity greater than 98% by TLC zonal scanning) (SA = 52.8 mCi/mmol) (5 tzCi) (The Radiochemical Centre, Amersham, UK) sterilized through a 0.22-/xm-diameter Millipore filter (Millipore Corp., Bedford, Massachusetts), was administered intravenously at 7 AM, ie, before breakfast, in saline solution. Total radioactivity administered was determined on 100 ~1 of the same solution. Six hours later a bile sample was collected by duodenal drainage after intravenous injection of cholecystokinin (CCK-PZ) (70 Ivy dog units/70 k g body weight) (Karolinska Institute, Sweden). Three ml of bile were retained, and the remainder returned into the duodenum. Further 3-ml bile samples were collected at the 24th, 48th and 72nd hours. Analytical Procedure. One ml of each bile sample was diluted in isopropanol (1:10, v/v), the remainder was stored at - 2 0 ~ C, A. Bile lipid composition: Total bile acid concentration was determined in each properly diluted bile sample by Sterognost-3c~ (Nyegard and Co-Ass, Oslo, Norway); p h o s p h o l i p i d s w e r e d e t e r m i n e d a c c o r d i n g to S v a m borg (12), cholesterol according to R6schlau's method (13) applied to bile by Roda et al (14), and the cholesterol saturation index (S1) according to Hegardt and Dam curves (15). B. Bile acid pool size and kinetics: Chenodeoxycholic acid pool size and kinetics were determined according to Linstedt's method (16); the pool size of other bile acid was calculated from the chenodeoxycholic acid pool size and from bile acid composition in bile assessed by gas-liquid chromatography (GLC), as described below. GLC Analysis. Four ml of each properly diluted bile sample were transferred into a Nickel bomb and dried under nitrogen stream. 8 ml of a mixture of 2 N N a O H and 50% methanol (1 : 1, v/v) were added to each sample. Hydrolysis was carried out at 120~ C for 4 hr; the hydrolyzed mixture was then quantitatively transferred into a large extraction tube. The bomb was washed with 2 ml (1 ml, 2• of deionized water and then added to the tube. The hydrolyzed mixture was finally acidified with 5 N HCl to pH = 1, and the bile acids were extracted three times with 30 ml of ethyl acetate. Radioactivity recovery was greater than 98%. The sample was dried under nitrogen stream and dissolved in 1 ml of chloroform-methanol (2:1, v/v); 50/xl of a 5.5 mg/ml of nordeoxycholic acid solution (kindly provided by Dr. A.F. Hofmann) was added as internal standard. Then 2 ml of ethereal diazomethane solution was added until the sample turned yellow. After 15 min, the sample was dried under nitrogen stream. The methylated bile acids were redissolved in 1 ml of chloroformmethanol (2 : 1, v/v), and the radioactivity was determined on 0.5 ml. Then 2 ml of the mixture of acetic anhydrideglacial acetic acid-perchloride acid (70%) (10:14:0.1, v/v/v) were added to the remainder in an ice bath.

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After standing at 40 C for 2 hr, 4 ml of a NaC1 (20%) solution was added to the sample, and bile acids were extracted three times with ethyl acetate and then reduced to dryness under nitrogen stream. The dried sample was redissolved in 0.1-0.3 ml of ethyl acetate and 0.2-1 /zl injected onto the column. A glass column (2 m x 0.3 cm) packed with 3% AN 600 on Anakrom Q (110-120 mesh) was used as follows: Column temperature Injector temperature Carrier gas Detector

260 ~ C 260 ~ C (all glass system) Nz 20-30 ml/min flame ionization

Qualitative analysis was based on the deoxycholic acid retention time. The variation of the molar concentration of the four bile samples was less than 2%, ranging from 0.6% for lithocholate to 1.5% for deoxycholate, 1% for chenodeoxycholate, 1.2% for ursodeoxycholate, and to 1.8% for cholate. Nordeoxycholic acid was used as internal standard for the quantitative analysis, correcting the areas according to the detector response. Radiochemical Analysis. A 0.5-ml sample of methylated bile was mn on a silica gel G plate (250/zm thickness, Merck, Darmstadt, GFR) together with each bile acid standard, using chloroform-acetone-methanol (70:20:5, v/v/v) as a solvent system (17). The Rf values for ursodeoxycholate and chenodeoxycholate were, respectively, 0.55 and 0.4. Chenodeoxycholic acid spots were scraped into scintillation vials. One ml of a mixture of ethanol-acetic acid (10: 1, v/v) was added. After mixing, 15 ml of Unisolve ~ was added to each vial. After dark equilibration at 4 ~ C, samples were counted in a Nuclear Chicago Isocap 300 scintillation spectrometer. Expression of Data. Calculation of parameters of bile acid kinetics was based on assumption of a first-order kinetics model, as described by Lindstedt (16). The logarithm of the specific activity of chenodeoxycholic acid, plotted vs time, was applied to the following formula: Logsr = Logso - bt (where: S = specific a c t i v i t y at t i m e t, t = t i m e , SO = specific activity at 0 time, b = slope) to determine the slope of the decay curve, which was always linear, as well as the specific activity at time 0. The pool of secondary bile acid was calculated from the measured pool of chenodeoxycholic acids and from the bile acid composition in bile, assessed by GLC. Intestinal metabolism, turnover rate, and half-life of the steroid were evaluated from the kinetics parameters. RESULTS

Effect of UDCA Administration on SI and Biliary Lipid Composition of Fasting Bile. P r i o r to t r e a t m e n t g a l l b l a d d e r bile w a s s u p e r s a t u r e d w i t h c h o l e s t e r o l ( T a b l e 1). A f t e r t r e a t m e n t , a significant d e c r e a s e ( P < 0.01) in SI a n d in the m o l a r p e r c e n t a g e o f c h o l e s t e r o l in bile w a s o b s e r v e d ( F i g u r e 1 a n d T a b l e 1).

Effect of UDCA Administration on Biliary Bile Digestive Diseases and Sciences, Vol. 24, No. 2 (February 1979)

URSODEOXYCHOLIC ACID ADMINISTRATION cholic acid (P

Effect of ursodeoxycholic acid administration on biliary lipid composition and bile acid kinetics in cholesterol gallstone patients.

Effect of Ursodeoxycholic Acid Administration on Biliary Lipid Composition and Bile Acid Kinetics in Cholesterol Gallstone Patients E N R I C O R O D...
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